GSDMD Antibody - #AF4013

FIGURE 5 The IL‐6R mAbs alleviated pyroptosis of THP‐1 by regulating NLRP3‐mediated inflammasome formation. (A) Immunoblotting and densitometry analysis of NLRP3, GSDMD, Caspase‐1 in the THP‐1 cells treated with the h‐mIL‐6R mAb (5 and 20 μg/ml) or Tocilizumab (5 μg/ml) for 30 min in advance, followed by treating with LPS (1 μg/ml) for 6 h. (B) ELISA and statistical analysis of IL‐1β levels in the supernatant of the THP‐1 cells with the indicated treatments identical to (A). (C,D) Flow cytometry analysis of cell pyroptosis with the indicated treatments in the THP‐1 cells. (E) Immunoblotting and densitometry analysis of NLRP3, GSDMD, Caspase‐1 in the THP‐1 cells treated with the h‐mIL‐6R mAb (10 μg/ml) or the NF‐κB inhibitor BAY 11–7082 (5 μM) for 30 min in advance, followed by treating with LPS (1 μg/ml) for 6 h. (F) ELISA and statistical analysis of IL‐1β levels in the supernatant of the THP‐1 cells with the indicated treatments identical to (E). (G,H) Flow cytometry analysis of cell pyroptosis with the indicated treatments in the THP‐1 cells. Cl‐Caspase‐1, cleaved Caspase‐1. The results were presented as mean ± SD through three independent experiments. The protein levels of N‐GSDMD and cleaved Caspase‐1 were normalized to the corresponding total proteins. The results were analyzed by one‐way ANOVA. *p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significance

Figure 2 BA attenuates pyroptosis after SCI. (A) Immunofluorescence staining for Caspase-1 and NeuN co-localization in the spinal cords of the Sham, SCI, and BA groups (scale bar = 25 µm) (B) The quantitative mean optical density of the Caspase-1 in motor neurons of spinal cord lesion. (C) Immunofluorescence staining for GSDMD and NeuN co-localization in the spinal cords of the Sham, SCI, and BA groups (scale bar = 25 µm) (D) The quantitative mean optical density of the GSDMD in motor neurons of spinal cord lesion. (E)Western blotting for ASC, Caspase-1, GSDMD, IL-1β, IL-18 and NLRP3 expression levels in the Sham, SCI, and BA groups. The gels were run under the same experimental conditions, and the cropped blots are shown here. (F) The optical density values of the ASC, Caspase-1, GSDMD, IL-1β, IL-18 and NLRP3 expression levels were quantified and analyzed in each group. The values are expressed as the means ± SEM, n=5 per group. **p< 0.01, vs. Sham group. #p< 0.05 and ##p< 0.01, vs. SCI group.

Figure 2 BA attenuates pyroptosis after SCI. (A) Immunofluorescence staining for Caspase-1 and NeuN co-localization in the spinal cords of the Sham, SCI, and BA groups (scale bar = 25 µm) (B) The quantitative mean optical density of the Caspase-1 in motor neurons of spinal cord lesion. (C) Immunofluorescence staining for GSDMD and NeuN co-localization in the spinal cords of the Sham, SCI, and BA groups (scale bar = 25 µm) (D) The quantitative mean optical density of the GSDMD in motor neurons of spinal cord lesion. (E)Western blotting for ASC, Caspase-1, GSDMD, IL-1β, IL-18 and NLRP3 expression levels in the Sham, SCI, and BA groups. The gels were run under the same experimental conditions, and the cropped blots are shown here. (F) The optical density values of the ASC, Caspase-1, GSDMD, IL-1β, IL-18 and NLRP3 expression levels were quantified and analyzed in each group. The values are expressed as the means ± SEM, n=5 per group. **p< 0.01, vs. Sham group. #p< 0.05 and ##p< 0.01, vs. SCI group.

FIGURE 3 CHBP inhibits pyroptosis in random-pattern skin flaps. (a–d) Skin tissue sections were stained with anti-GSDMD-N (green) or anti-caspase 1 (CASP1; red) immunofluorescence antibodies (DAPI staining of the nuclei). Scale bar: 10 μm. The number of positive cells is shown on the right of representative images (n = 6 mice per group). (e,f) CHBP treatment reduced the expression of NLRP3, ASC, caspase 1 (CASP1), GSDMD-N, IL-1β and IL-18 in the context of ischaemia–reperfusion injury. Densitometric quantification is shown on the right (n = 6 mice per group). (g–j) CHBP treatment attenuated the activation of GSDMD, IL-1β, IL-18 and caspase 1 in the skin tissues detected by ELISA kits (n = 6 mice per group). Data shown are means ± SEM. *P 

FIGURE 3 CHBP inhibits pyroptosis in random-pattern skin flaps. (a–d) Skin tissue sections were stained with anti-GSDMD-N (green) or anti-caspase 1 (CASP1; red) immunofluorescence antibodies (DAPI staining of the nuclei). Scale bar: 10 μm. The number of positive cells is shown on the right of representative images (n = 6 mice per group). (e,f) CHBP treatment reduced the expression of NLRP3, ASC, caspase 1 (CASP1), GSDMD-N, IL-1β and IL-18 in the context of ischaemia–reperfusion injury. Densitometric quantification is shown on the right (n = 6 mice per group). (g–j) CHBP treatment attenuated the activation of GSDMD, IL-1β, IL-18 and caspase 1 in the skin tissues detected by ELISA kits (n = 6 mice per group). Data shown are means ± SEM. *P 

Figure 6 BA alleviates pyroptosis and inflammation in the spinal cord tissue of SCI rats. On day 18 after SCI, Western blot assay was performed. (A–D) NLRP3 (A), ASC (B), GSDMD (C), and Caspase-1 (D) mRNA expression levels were measured by quantitative reverse transcription-polymerase chain reaction. (E) Western blot analysis of inflammation- and pyroptosis-associated protein expression levels. β-Actin was used as an internal reference for NLRP3, ASC, GADMD, IL-18, and IL-1β. GAPDH was used as an internal reference for TLR4, P65, p-P65, IκBα, and p-IκBα. (F–M) NLRP3 (F), ASC (G), GSDMD (H), IL-18 (I), IL-1β (J), TLR4 (K), p-P65/P65 (L), and p-IκBα/IκBα (M) protein expression levels were measured by Western blot assay. All western blot data were normalized to the sham group. Data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, vs. sham group; #P < 0.05, ##P < 0.01, vs. model group (one-way analysis of variance followed by Tukey's post hoc test). ASC: Apoptosis-associated speck-like protein containing CARD; BA: Biochanin A; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GSDMD: gasdermin D; IL-18: interleukin-18; IL-1β: interleukin-1β; NLRP3: NOD-like receptor thermal protein domain associated protein 3; PC: positive control; p-P65: phospho-NF-κB P65; p-IκBα: phospho-NF-kappa-B inhibitor alpha; SCI: spinal cord injury; TLR4: Toll-like receptor 4.

Figure 3 Bexarotene attenuates neuronal pyroptosis in the spinal cord after SCI. (A) Immunofluorescence staining of Caspase 1 (pyroptosis-related marker, red) and NeuN (indicating neurons, green) (original magnification 30×) in spinal cord sections. The optical densities of Caspase 1 were markedly decreased in the SCI + Bex group and significantly increased in the SCI group. Scale bars: 25 μm. (B) Quantification of Caspase-1 in neurons of spinal cords in A. (C) Immunofluorescence staining of GSDMD-N (pyroptosis-related protein, green) and NeuN (indicating neurons, red) (original magnification 30×). Scale bars: 25 μm. (D) Quantification of GSDMD in neurons of spinal cords in C. (E–H) Evaluation of CASP1, GSDMD, IL-18, and IL-1β in the spinal cord by ELISA. (I) Western blot assay of GSDMD-N, NLRP3, Caspase-1, IL-1β, IL-18, and ASC. (J) Quantitative analyses of data from I; data were normalized to GAPDH. Data are expressed as the mean ± SEM (n = 6 mice per group). **P < 0.01, vs. sham group; ##P < 0.01, vs. SCI group (one-way analysis of variance with the least significance difference post hoc test). ASC: Apoptosis-associated speck-like protein containing a CARD; Bex: bexarotene; C-CASP-1: cleaved Caspase 1; DAPI: 4′,6-diamidino-2-phenylindole; ELISA: enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde-3- phosphate dehydrogenase; GSDMD-N: gasdermin D-N; IL: interleukin; IOD: integrated optical density; NLRP3: NOD-like receptor thermal protein domain associated protein 3; SCI: spinal cord injury.

Figure 3 Bexarotene attenuates neuronal pyroptosis in the spinal cord after SCI. (A) Immunofluorescence staining of Caspase 1 (pyroptosis-related marker, red) and NeuN (indicating neurons, green) (original magnification 30×) in spinal cord sections. The optical densities of Caspase 1 were markedly decreased in the SCI + Bex group and significantly increased in the SCI group. Scale bars: 25 μm. (B) Quantification of Caspase-1 in neurons of spinal cords in A. (C) Immunofluorescence staining of GSDMD-N (pyroptosis-related protein, green) and NeuN (indicating neurons, red) (original magnification 30×). Scale bars: 25 μm. (D) Quantification of GSDMD in neurons of spinal cords in C. (E–H) Evaluation of CASP1, GSDMD, IL-18, and IL-1β in the spinal cord by ELISA. (I) Western blot assay of GSDMD-N, NLRP3, Caspase-1, IL-1β, IL-18, and ASC. (J) Quantitative analyses of data from I; data were normalized to GAPDH. Data are expressed as the mean ± SEM (n = 6 mice per group). **P < 0.01, vs. sham group; ##P < 0.01, vs. SCI group (one-way analysis of variance with the least significance difference post hoc test). ASC: Apoptosis-associated speck-like protein containing a CARD; Bex: bexarotene; C-CASP-1: cleaved Caspase 1; DAPI: 4′,6-diamidino-2-phenylindole; ELISA: enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde-3- phosphate dehydrogenase; GSDMD-N: gasdermin D-N; IL: interleukin; IOD: integrated optical density; NLRP3: NOD-like receptor thermal protein domain associated protein 3; SCI: spinal cord injury.

Figure 3 Bexarotene attenuates neuronal pyroptosis in the spinal cord after SCI. (A) Immunofluorescence staining of Caspase 1 (pyroptosis-related marker, red) and NeuN (indicating neurons, green) (original magnification 30×) in spinal cord sections. The optical densities of Caspase 1 were markedly decreased in the SCI + Bex group and significantly increased in the SCI group. Scale bars: 25 μm. (B) Quantification of Caspase-1 in neurons of spinal cords in A. (C) Immunofluorescence staining of GSDMD-N (pyroptosis-related protein, green) and NeuN (indicating neurons, red) (original magnification 30×). Scale bars: 25 μm. (D) Quantification of GSDMD in neurons of spinal cords in C. (E–H) Evaluation of CASP1, GSDMD, IL-18, and IL-1β in the spinal cord by ELISA. (I) Western blot assay of GSDMD-N, NLRP3, Caspase-1, IL-1β, IL-18, and ASC. (J) Quantitative analyses of data from I; data were normalized to GAPDH. Data are expressed as the mean ± SEM (n = 6 mice per group). **P < 0.01, vs. sham group; ##P < 0.01, vs. SCI group (one-way analysis of variance with the least significance difference post hoc test). ASC: Apoptosis-associated speck-like protein containing a CARD; Bex: bexarotene; C-CASP-1: cleaved Caspase 1; DAPI: 4′,6-diamidino-2-phenylindole; ELISA: enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde-3- phosphate dehydrogenase; GSDMD-N: gasdermin D-N; IL: interleukin; IOD: integrated optical density; NLRP3: NOD-like receptor thermal protein domain associated protein 3; SCI: spinal cord injury.

FIGURE 4 | Baicalein attenuates pyroptosis after spinal cord ischemia-reperfusion injury (SCIR).(C) Western blotting for NLRP3, GSDMD, and C-CASP1 expression levels in the Sham, SCIR+Vehicle, and SCIR+Baicalein groups. The gels were run under the same experimental conditions, and the cropped blots are shown here.